High-throughput crystallographic screening device and method for crystalizing membrane proteins using a sub physiological resting membrane potential across a lipid matrix of variable composition

ABSTRACT

The invention is a high-throughput voltage screening crystallographic device and methodology that uses multiple micro wells and electric circuits capable of assaying different crystallization condition for the same or different proteins of interest at the same of different voltages under a humidity and temperature controlled environment. The protein is solubilized in a lipid matrix similar to the lipid composition of the protein in the native environment to ensure stability of the protein during crystallization. The invention provides a system and method where the protein is transferred to a lipid matrix that holds a resting membrane potential, which reduces the degree of conformational freedom of the protein. The invention overcomes the majority of the difficulties associated with vapor diffusion techniques and essentially reconstitutes the protein in its native lipid environment under “cuasi” physiological conditions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/996,946, filed Jun. 4, 2018 now U.S. Pat. No. 11,440,001, which is aContinuation of U.S. patent application Ser. No. 15/638,276, filed Jun.29, 2017 now abandoned, which claims the benefit of priority under 35U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62/516,860,filed Jun. 8, 2017, and to U.S. Provisional Patent Application No.62/356,266, filed Jun. 29, 2016. The disclosures set forth in thereferenced applications are incorporated herein by reference in theirentireties.

GOVERNMENT INTEREST

The claimed invention was made with U.S. Government support under grantnumber R01 GM098343 awarded by the National Institutes of Health (NIH).The government has certain rights in this invention.

BACKGROUND

Approximately 30% of the human genes code for membrane proteins. Despitethe efforts made by the best worldwide crystallographers, only minutefraction of the entries in the Protein Data Bank correspond to membraneproteins. A 3D protein structure is critical to the advancement andefficiency of rational drug design, as well as to proteinstructure-function studies, because the majority of drugs and naturaleffector molecules stereo-specifically interact with target proteins toaffect their physiological and biological activity by blocking oraltering its properties.

Membrane proteins have hydrophobic domains and are expressed atrelatively low levels. This creates difficulties in obtaining enoughprotein and growing crystals. The determination of high-resolutionstructures for these proteins is far more difficult than globularproteins. Nowadays, less than 0.1% of protein structures determined aremembrane proteins.

The crystallization process completely depends on the organizationability of the proteins in a medium. Once these proteins are organizedrepetitively in a solid three-dimensional lattice, it is that thecrystal of the protein is formed. This process is regulated byphysical-chemical, kinetic and thermodynamic factors and consists of twosteps. The first step is known as nucleation, in which the proteinmolecules that are dissolved in the matrix originally used to collect itfrom their natural environment, begin to cluster. This gives rise to anextremely small focus, nucleus, on the solution where there is a higherconcentration of the protein as a solute. The second step is thecontinuous and orderly growth of this small focus of crystals.Nucleation can be initiated by the inclusion of a precipitating agent asis the case in the vapor diffusion technique.

During these processes the proteins could diffused and grouped accordingto the conformation that it acquires both in the extraction matrix usedfor its production and in the medium in which it is being precipitated.Therefore, the crystals that form, if this occurs, do not necessarilyreflect the “true” structure of these proteins in their naturalenvironment. Specifically, many technical problems are associated withthe task of membrane protein crystallization. The principal problem withthe crystallization of membrane proteins is that they are difficult tohandle and solubilize from its native environment in such a way thatretains native conformation and activity. Then, the solubilizedprotein-detergent complex needs to be placed in an environment similarto the native membrane and force nucleation. Membrane proteins areinherently amphiphilic, they comprise hydrophobic and hydrophilicregions. Due to their amphiphilic nature, membrane proteins tend toaggregate rapidly to minimize the hydrophobic regions. The addition ofprecipitants often causes an interaction with the solubilizedprotein-detergent complex that induces phase separation. For severaldecades the crystallization of membrane proteins has been done usingvapor diffusion methods including hanging drop and sitting drop. Themajority of the crystallization methods using vapor diffusion techniquesrely on reducing the solubility of proteins in an aqueous environment,for instance isoelectric focusing methods.

All membrane proteins are embedded inside a lipid membrane that holds aresting membrane potential (RMP). On the basis of this fundamentalprinciple, we believe that the structural conformations of membraneproteins (including ligand gated channels) are voltage-dependent. Themost remarkable example for the voltage-dependent conformation of aprotein is the large family of voltage-dependent ion channels. Our groupfurther studied this concept while recoding single channel currents(cell-attached) in myocytes. In order to estimate the opening andclosing rate constants (at −80 mV), it was necessary to record at least100 bursts per acetylcholine concentration [ACh]. At high AChconcentrations (>500 μM) the number of bursts per [ACh] was dramaticallyreduced as a result of desensitization. To overcome this problem, wemade a quick change in the polarity of the amplifier (from −80 mV to +80mV and back to −80 mV in ˜1 sec) and the single burst activity recoveredimmediately. This experiment revealed that at +80 mV the agonist wasexpelled from the ACh binding site and the channel conformation shiftedfrom the desensitized conformation and immediately equilibrated betweenthe open and closed states until it desensitized again. Thus, even in aligand-gated channel such as the nAChR, the desensitized conformationcan be reversed by changing the RPM. The biophysical principle here isthat a membrane protein sits in a voltage gradient across a membrane andsome localized domains in the protein can display voltage dependency.

Accordingly, what is needed is a system and a method for thecrystallization of membrane proteins without the limitations andconstraints of the prior art systems and techniques including vapordiffusion methods and LCP.

SUMMARY

The invention is a high-throughput voltage screening crystallographicdevice and methodology for protein crystallization which consists ofthree layers of multiple micro wells electric circuit capable ofassaying different crystallization condition for the same or differentproteins of interest at different voltages under a humidity andtemperature controlled environment.

The methodology of the invention could be used with a singlemicro-capillary crystal tube of different internal diameters pre-cut foreasy recovery of the protein crystal, or it can be configured to allowmultiple crystallization conditions in parallel.

According to an aspect of the invention, the crystallization systemenables the use of close amphiphilic environments (e.g. monooelein) formembrane protein crystallization and the rates of evaporation arecontrolled by the relative humidity conditions, which are adjusted in aprecise and stable way during the combination of the solubilizedprotein-detergent complex and amphiphilic reagents.

According to another aspect of the invention, the protein crystals cannucleate and grow under different dehydration conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the office upon request and paymentof the necessary fee.

Further features and advantages of the invention will become apparentfrom the following detailed description taken in conjunction with theaccompanying figure showing illustrative embodiments of the invention,in which:

FIG. 1 illustrates the components of the sample unit according to anembodiment of the present invention.

FIG. 2 illustrates the sample unit partially assembled according to anembodiment of the present invention.

FIG. 3 illustrates the sample unit assembled according to an embodimentof the present invention.

FIG. 4 illustrates a side cross-sectional view of the sample unitaccording to an embodiment of the present invention.

FIG. 5 illustrates a sample unit according to another embodiment of thepresent invention.

FIG. 6 illustrates a crystallization system according to an embodimentof the present invention.

FIG. 7 illustrates the sample unit preparation for a standard proteinsample loading according to an embodiment of the present invention.

FIG. 8 illustrates the sample unit preparation for a vapor diffusionprotein sample loading according to an embodiment of the presentinvention.

FIG. 9 shows images of protein samples without applying electricpotential.

FIG. 10 shows images of protein crystals for a fluorescence-labeledprotein according to an embodiment of the present invention.

FIG. 11 shows images of a plurality of nAChR crystals inside the sampleunit according to an embodiment of the present invention.

FIG. 12 shows images of a plurality of nAChR crystals inside the sampleunit according to an embodiment of the present invention.

FIG. 13 shows a confocal microscopy image of a nAChR-αBTX crystal in thesample unit according to an embodiment of the present invention.

FIG. 14 shows an image of a nAChR crystal in the loop according to anembodiment of the present invention.

FIG. 15 a shows an image of a nAChR crystal in the sample unit accordingto an embodiment of the present invention.

FIG. 15 b shows an image of a nAChR crystal in the loop according to anembodiment of the present invention.

FIG. 16 a shows an image of a nAChR crystal in the sample unit accordingto an embodiment of the present invention.

FIG. 16 b shows images of a nAChR crystal in the loop according to anembodiment of the present invention.

FIG. 17 shows histograms for protein crystal formation frequencydistribution according to an embodiment of the present invention.

FIG. 18 shows images of protein crystallization using Voltage-LipidicCubic Phase-Fluorescence Recovery After Photobleaching techniqueaccording to an embodiment of the present invention.

FIG. 19 shows fractional fluorescence recovery and mobile fraction plotsfor a Fluorescence Recovery After Photobleaching Assay according to anembodiment of the present invention.

Throughout the figures, the same reference numbers and characters,unless otherwise stated, are used to denote like elements, components,portions or features of the illustrated embodiments. The subjectinvention will be described in detail in conjunction with theaccompanying figures, in view of the illustrative embodiments.

DETAILED DESCRIPTION

The system of present invention provides a sample unit 1 including asample holding layer 1 a and a lid layer 1 b, wherein the sample holdinglayer 1 a includes at least one well 1 c with a pair of electrodes 1 dand the lid layer 1 b also includes at least one well 1 e as shown inFIGS. 1-4 . Alternatively, depending on the protein loading method thelid layer can be substituted with thin strip of clear adhesive tape.According to a preferred embodiment of the invention, the volume perwell in the sample holding layer is 50 μl and 25 μl in the lid layerwell. However, other volumes can also be used depending on severalfactors or parameters such as but no limited to the amount of sampleneeded. As can be appreciated, the wells have a rectangular shape withthe dimensions being selected based on the volume desired to hold thesample for crystallization purposes. In accordance to anotherembodiment, the wells are provided as round-shaped wells (FIG. 5 ) withthe dimensions also being selected based on the volume desired to holdthe sample for crystallization purposes. One advantage of usinground-shaped wells is that smaller-volume wells can be provided as theelectrodes can be positioned closer to each other requiring smallervoltages and increasing the magnetic field effect duringcrystallization.

FIG. 6 illustrates a general crystallization system according to thepresent invention. An incubator 2 is provided to incubate the sampleunit 1 during the crystallization process. A power supply unit 3 isconnected to the electrodes of the sample holding layer 1 a to providethe required voltage in accordance with the invention. In addition, atemperature and humidity monitoring means 4 is provided to monitor andcontrol the crystallization process within specific temperature andhumidity conditions. Other monitoring and control means can be providedto ensure proper crystallization of the proteins in accordance to themethod of the present invention. All these components can be providedexternally to the incubator 2 or alternatively can be integrated as partof the incubator 2.

One important advantage of the invention is that multiple sample unitscan be incubated simultaneously while applying a desired voltage andmaintaining specific incubation conditions. For example, differentprotein concentrations can be provided in different sample units or indifferent wells of the same sample unit. Also, the system of theinvention allows to supply the same voltage to all the wells of thesample units or different voltages can be supplied to each well. Inaddition, with proper control and monitoring different crystallizationtechniques could be simultaneously carried out in the incubator. Thevoltage supplied to the sample units can be provided by a single voltagesource or via a multi-voltage source. This can be done using pluralregulated voltage sources or using a multi-voltage regulated outputcircuits. It is important to note that the selection of regulatedvoltage sources as well as the voltage ranges will depend on the type ofprotein and membrane resistance which determines the range of membranepotentials in which membrane protein crystals are formed in a definedlipid matrix composition also using different electrode diameters. Inaddition, the resistance of the lipid membrane is critical to assurethat ion flux is constant during the crystallization process. Our datashows that crystallization of membrane proteins can occur within a verylimited range of sub membrane potentials. As can be appreciated, thesystem of the present invention is a highly-configurable and flexiblesystem that can be used with different protein crystallization methodsand overcomes the majority of the difficulties associated with thetypical methods.

In operation, the protein of interest needs to be extracted, purifiedand properly prepared prior to loading into the sample unit 1. Note thatthis step will vary depending on several factors including but notlimited to: the type and amount of protein, physiological pH of theprotein, ionic strength of the medium, optimal crystallizationpotential, and solubility of the detergent among others. For example,according to an embodiment of the invention, Nicotinic AcetylcholineReceptor (nAChR) extraction was performed by homogenizing 200 g ofTorpedo californica tissue. To perform the solubilization of the crudemembranes, they were thawed, and mixed with a 1% detergent solutioncontaining DB-1× Buffer (100 mM NaCl, 10 mM MOPS, 0.1 mM EDTA, and 0.02%NaN3). The detergent used to extract the transmembrane proteins wasLysoFos Choline 16, Anagrade (LFC-16). After extraction, purificationstep was carried out using affinity chromatography. During the column'spreparation, Bromoacetylcholine bromide was coupled to Affigel 10(Bio-Rad) with DB-1× as a coupling buffer. The first step of thepreparation of Affigel-10 was to incorporate sulfhydryl groups. To doso, 25 ml of Affigel-10, to which the conservator was eliminated througha series of washes in isopropanol and water, was equilibrated with 50 mlof 20 mM MOPS at pH 7.4. Afterwards 50 ml of cysteine 0.054 M was added,allowing it to react for one hour. After the cysteine excess was rinsedoff with 200 ml of water, 50 ml of the reducing agent dithiothreitol(DTT) 0.1 M with MOPS at pH 8.0 was added for thirty minutes. Afterequilibration using 100 ml of water, 500 mg of Bromoacetylcholinebromide was added, which attached to the thiol groups in the gel. Theremaining thiol groups were blocked with 50 mg of iodoacetamide. Oncethe Affigel-10 had undergone anhydrous coupling, it was placed in anEcono Bio-Rad 1.5×20 cm column and stored at 4° C. with a low ionicforce of 50 mM Sodium Acetate pH 4.0. The solubilized extracts of crudemembranes were passed through the column, during which the nAChRsattached to the acetylcholine by affinity. The elution of thechromatographic matrix receptor was performed with a solution containingcarbamylcholine, which has greater affinity in the column. This yieldsan elution solution containing purified nAChRs. All steps were carriedout in the cold room (4° C.) or keeping the samples on ice. As can beunderstood, one skill in the art would know the exact conditions andparameters for protein extraction and purification that would providethe optimal conditions for crystal formation in accordance with thepresent invention.

Once the protein has been prepared, it needs to be loaded into thesample units 1 prior to placement inside the incubator 2. This step willvary depending on the type of crystallization desired. FIG. 7illustrates the steps for a standard protein loading where thecrystallization precipitant solution is added to each well 1 c of thesample holding layer 1 a and the protein sample is later deposited inthe center of each well 1 c using a dispenser between the circuitelectrodes ensuring contact between the electrodes. Afterwards, thesample unit is sealed with the lid layer 1 b or alternatively a layer ofclear adhesive tape. For a vapor diffusion protein loading, as shown inFIG. 8 , the protein sample is deposited in the center of each well 1 cof the sample holding layer 1 a between the circuit electrodes 1 d,wherein the protein sample must be in lipid cubic phase and must be incontact with both circuit electrodes 1 d. Then, the crystallizationprecipitant solution is added to each well 1 e of the lid layer 1 b andfinally the the sample holding layer 1 a is turned over or flipped 180°and placed on top of the lid layer 1 b to seal the sample unit. It isimportant to point out that the incubator 2 should be at the desiredtemperature prior to loading the proteins in the sample units.

The next step is to calibrate and prepare the system for incubationduring the crystallization process. To that effect, the voltage sourceis turned on and adjusted to the maximum voltage value to be applied tothe sample units and then while measuring the voltage, adjusting theoutput voltage to the desired values. Afterwards, the voltage source isturned off and its output is electrically connected to the electrodesarrangement on the sample units inside the incubator 2. The voltage oneach well Is measured and adjusted accordingly to ensure the requiredvoltage for crystallization, wherein the incubator is finally closedwith the sample units inside ready for crystallization.

Finally, the proteins are incubated for a predetermined amount of time,which according to a preferred embodiment of the invention is between1-2 weeks. When the crystallization process is finished the voltagesupply is turned off and disconnected from the sample units holding theprotein crystals for subsequent removal from the incubator and crystalextraction for appropriate analysis.

There are several aspects of the system and methodology to consider whenusing the present invention for protein crystallization. First, themembrane protein sample to be crystallized in this system must be highlypure to ensure optimal crystallization. Also, the membrane protein issolubilized in a lipid matrix of variable composition at a particularlipid to protein ratio and to ensure stability of the membrane protein,the lipid composition used for the crystallization must be similar tothe lipid composition of the protein in the native environment. To thateffect, a lipidomic analysis of the model membrane protein must beperformed and a lipid matrix containing lipid-detergent analogs similarto the native lipid composition of the protein must be used. Inaddition, a variety of lipid phases can be used with the invention,which in turn results in a variable degree of hydration. Furthermore,the lipid composition of the matrix can be variable depending on thetype of membrane protein sample. In an embodiment of the invention, theresistance of the lipid matrix is in the range of 1-100 MΩ. However,other ranges of resistances such as 25-200 MΩ can be used depending onthe protein size, protein concentration and lipid composition. It isimportant to point out that the resistance of the lipid matrix (LMx)remains variable in the initial phases of the crystallization, however,it must reach a constant value during the crystallization procedure(24-168 hours). Optionally, at any given point during thecrystallization procedure, lipid doping can be performed depending onthe resistance of the lipid-protein matrix and the membrane protein.Furthermore, a variable physiological membrane potential (−140 mv-10 mV)can be used to stabilize the membrane protein conformation at thebeginning of the experiment and after a period of 1-2 hours thepotential can be slowly decreased to reach a sub physiological range ofpotential (−5 mV to −20 mV) where it can be either kept constant orchanged (voltage-ramp mode) for the remaining period of thecrystallization process. In addition, the pH and ionic content of thelipid matrix can be manipulated during crystallization. Membrane proteincrystal formation occurs in a time frame of 24-96 hours depending on themembrane protein concentration and composition of the lipid matrix andthe crystals are produced at room temperature or, if necessary, at lowertemperatures. Also, lipid diffusion experiments can be performed tooptimize crystal formation and quality.

An important feature of the invention is that when using a fluorescenttagged membrane protein the system will allow monitoring crystalformation and membrane protein stability during crystallization process.Moreover, Fluorescence Recovery After Photobleaching (FRAP) experimentscan be used during the crystallization process to determine mobilefraction of the membrane protein and to optimize the lipid compositionof the lipid matrix to achieve crystallization. It is important to pointout that mobile fraction of membrane proteins in the lipid matrix willhave to be over 75% to facilitate crystallization. Another importantfeature of the invention is that the system allows performing X-raydiffraction experiments in situ and that there is no limitation in themolecular weight (MW) of the protein, thus larger membrane complexes canbe crystallized. This is extremely important since the inventionovercomes the MW weight limitation that is intrinsic to the LipidicCubic Phase (LCP) methodology. The membrane protein crystal is harvestedwhile the protein is grown at a sub-physiological membrane potential (−5mV to −140 mV) and the membrane protein crystal is immediately frozen at−80° C.

The effectiveness of the present invention will be now explained inaccordance to FIGS. 9-19 . FIG. 9 shows images of samplescrystallization without applying electric potential according to thepresent invention. As can be appreciated, no well-formed crystals werefound. In clear contrast FIG. 10 shows the formation of proteincrystals, where nAChR is conjugated with a-Bungarotoxin Alexa Fluor® 488for fluorescence and was prepared using a lipid cubic phase (LCP)technique (the arrows indicate the crystal formation). FIG. 11 showsseveral nAChR crystals growing in different sizes inside the sample unitaccording the present invention. FIG. 12 shows nAChR crystals formedfrom samples that were conjugated with α-BTX and monoclonal antibodies.FIG. 13 shows a confocal microscopy image of a well formed nAChR-αBTXcrystal in the sample unit, where the crystal structure can beappreciated. FIG. 14 shows an image of a nAChR crystal (indicated by thearrow) in the loop ready to be diffracted. FIGS. 15 a and 15 b showimages of a nAChR crystal in the sample unit (indicated by the arrow)and in the loop ready to be diffracted, respectively. FIGS. 16 a and 16b show additional images of the nAChR crystal in the sample unit(indicated by the arrow) and in the loop ready to be diffracted,respectively. FIG. 17 shows histograms (for raw and normalized data) ofprotein crystal formation frequency distribution, where differentvoltages where applied for the stimulation of crystal nucleation and thesample size for the experiment was 160. FIG. 18 shows images ofVoltage-Lipidic Cubic Phase-Fluorescence Recovery After PhotobleachingAssay (V-LCP-FRAP) using a lipid cubic phase (LCP) in which the sampleis placed in a lipidic and viscous environment. The Region of Interest(ROI) are the areas in which fluorescence recovery is measured. Thisassay was performed in order to determine which protein-detergentcomplexes provide the highest protein stability, for structural studies.FIG. 19 shows fractional fluorescence recovery and mobile fractiongraphs for a FRAP assay experiment where lipidic cubic phase (LCP) wasused. This experiment was carried out with the implementation ofmonoclonal antibodies in monoolein matrix for the nAChR-α-BTX complexusing phospholipid analog detergent, where all experiments wereperformed in triplicate and the incubation was 20° C. and recorded everyfive days (three times for mAb-F8 and one time for mAb-B2).

A fundamental aspect of the present invention is the principle ofmembrane resistance. To this effect, the conditions for membrane proteincrystal formation were assessed using a basic electrode prototype todetermine the range of membrane potentials in which membrane proteincrystals are formed in a defined lipid matrix composition usingdifferent electrode diameters. In a lipid matrix of define composition,crystal formation was observed within a resistance range of 1-25 MΩdepending on the protein concentration in the lipid matrix. Theresistance range also depends on the size and molecular weight of theproteins because these are intrinsic parameters that affect the membranecapacitance. The resistance of the lipid membrane is critical to assurethat ion flux is constant during the crystallization process. Our datashows that crystallization of membrane proteins can occur within a verylimited range of sub membrane potentials.

The present invention overcomes the majority of the difficultiesassociated with vapor diffusion techniques (i.e, hanging drop, sittingdrop, etc.), because the protein-detergent complex is rapidly mixed witha lipid matrix (LMx) of defined composition. Second, the detergent isimmediately diluted in an enriched lipid matrix (LMx) where it diffusesfrom the protein-detergent complex in a native hydrophobic/aqueousenvironment allowing critical lipid-protein (and van Der Waals')interactions with the hydrophobic domains of the membrane protein.Third, the dilution and diffusion of the detergent from theprotein-detergent complex under the aforementioned conditions iscritical to preserve stability of the membrane protein and to reduce theaggregation caused by denaturation of protein hydrophobic domains.Fourth, during the process of detergent diffusion the membrane proteinis presumably preserved in a single conformation by the membranepotential in LMx with constant resistance. Lastly, this methodologyessentially reconstitutes the membrane protein in its native lipidenvironment under “cuasi” physiological conditions.

It is important to emphasize that the present invention provides asystem and method where the membrane protein is transferred to a lipidmatrix that holds a resting membrane potential, which reduces the degreeof conformational freedom of the protein. The system and methodology ledto consistent x-Ray diffractions from the nAChR-LFC16 complex. Theinvention will serve to test new approaches in a very challenging fieldof structural Biology and it represents a step forward in the use ofinnovative approaches for the solution membrane protein structures. Thismethodology was developed after many attempts to crystallize the nAChRusing vapor diffusion methods and, more recently, LCP. Our team hasbeing doing electrophysiological recordings of nAChR channel activityfor many years and our basic understanding of the nAChR structure andfunction was conceptualized in a physiological environment. The systemof the present invention was conceived to crystalize the nAChR in itsclosest physiological environment, which includes a native lipidcomposition and a fixed resting membrane potential.

Although the invention has been described in conjunction with specificembodiments, it is evident that many alternatives and variations will beapparent to those skilled in the art in light of the foregoingdescription. Accordingly, the invention is intended to embrace all ofthe alternatives and variations that fall within the spirit and scope ofthe invention.

What is claimed is:
 1. A system for membrane protein crystallizationcomprising: a sample unit having: a sample holding layer having aplurality of holding wells, wherein each holding well comprises a pairof electrodes, and a lid layer provided over said plurality of holdingwells to seal the contents of said plurality of holding wells from eachother; and a variable direct current potential source electricallycoupled to the pair of electrodes of each holding well.
 2. The system ofclaim 1, further comprising: an incubator receiving the sample unit; atemperature unit monitoring and controlling a temperature inside theincubator; and a humidity unit monitoring and controlling a humidityinside said incubator.
 3. The system of claim 1, wherein the variablepotential source is configured to alter a direction of a potential fieldgenerated between the pair of electrodes of each holding well, whereinthe direction of the potential field generated varies within 180degrees.
 4. The system of claim 1, wherein the plurality of holdingwells has the same geometric shape or different geometric shapes.
 5. Thesystem of claim 1, wherein said lid layer comprises a plurality of lidwells having the same shape and dimensions as said holding wells.
 6. Thesystem of claim 1, wherein the pair of electrodes has the same geometricshape in every holding well.
 7. The system of claim 1, wherein at leastone holding well has a pair of electrodes having a geometric shapedifferent than the pair of electrodes ends of said plurality of holdingwells.
 8. The system of claim 1, wherein said variable direct currentpotential source provides the same variable direct current potential tothe pair of electrodes ends of all holding wells.
 9. The system of claim1, wherein said variable direct current potential source providesdifferent variable direct current potentials to the pair of electrodesends of different holding wells.
 10. The system of claim 1, wherein saidsample unit comprises at least one membrane protein sample, and whereinsaid at least one membrane protein sample comprises solubilized membraneprotein complex.
 11. The system of claim 10, wherein said at least onemembrane protein sample is provided in a lipid matrix.
 12. The system ofclaim 11, wherein said lipid matrix has a lipid composition similar tothe lipid composition of the membrane protein in the native environmentor is varied by lipid doping.
 13. The system of claim 1, wherein saidsample unit comprises a plurality of membrane protein samples of thesame membrane protein.
 14. The system of claim 1, wherein said sampleunit comprises a plurality of membrane protein samples of differentmembrane proteins.
 15. The system of claim 1, wherein said potentialsource provides to the pair of electrodes ends of the holding wells apotential selected from: a subphysiological membrane potential, aphysiological membrane potential and a supra-physiological membranepotential.
 16. The system of claim 1, wherein said potential sourcevaries a waveform of said potential.
 17. A system for membrane proteincrystallization comprising: a sample unit having: a sample holding layerhaving a plurality of holding wells, wherein each holding well comprisesa pair of electrodes, and a lid layer provided over said plurality ofholding wells to seal the contents of said plurality of holding wellsfrom each other; a variable direct current potential source electricallycoupled to the pair of electrodes of each holding well; wherein thevariable potential source is configured to alter the intensity of apotential field generated between the pair of electrodes of each holdingwell.
 18. The system of claim 17, wherein the alteration in intensity ofa potential field occurs at a range of potential from −140 mV to 10 mV.19. The system of claim 17, wherein the variable potential source isconfigured to alter a direction of a potential field generated betweenthe pair of electrodes of each holding well, wherein the direction ofthe potential field generated varies within 180 degrees.
 20. The systemof claim 17, wherein a temperature unit and a humidity unit control thetemperature and humidity conditions during a crystallization process ofa membrane protein sample contained within a holding well of said sampleunit.
 21. The system of claim 20, wherein at least one of: the potentialsource, the temperature unit and the humidity unit is integrated into anincubator.
 22. The system of claim 17, wherein said lid layer comprisesa plurality of lid wells having the same shape and dimensions as saidholding wells.
 23. The system of claim 17, wherein said variable directcurrent potential source provides the same variable direct currentpotential to the pair of electrodes ends of all holding wells.
 24. Thesystem of claim 18, wherein said sample unit comprises at least onemembrane protein sample, and wherein said at least one membrane proteinsample comprises solubilized membrane protein complex.
 25. A system formembrane protein crystallization comprising: a sample unit having: asample holding layer having a plurality of holding wells, wherein eachholding well comprises a pair of electrodes, and a lid layer providedover said plurality of holding wells to seal the contents of saidplurality of holding wells from each other; a variable direct currentpotential source electrically coupled to the pair of electrodes of eachholding well; wherein the variable potential source is configured toalter the intensity of a potential field generated between the pair ofelectrodes of each holding well more than one hour after the firstvariation in the potential field.
 26. The system of claim 25, whereinthe potential is altered to a range from −5 mV to −20 mV after 1-2 hoursin a range from −140 mV to 10 mV.
 27. The system of claim 25, whereinthe variable potential source is configured to alter a direction of apotential field generated between the pair of electrodes of each holdingwell, wherein the direction of the potential field generated varieswithin 180 degrees.
 28. The system of claim 25, wherein said sample unitcomprises a plurality of membrane protein samples of the same membraneprotein.
 29. The system of claim 25, wherein said sample unit comprisesa plurality of membrane protein samples of different membrane proteins.30. The system of claim 25, wherein said potential source provides tothe pair of electrodes ends of the holding wells a potential selectedfrom: a subphysiological membrane potential, a physiological membranepotential and a supra-physiological membrane potential.
 31. The systemof claim 25, wherein said sample unit comprises at least one membraneprotein sample, and wherein said at least one membrane protein samplecomprises solubilized membrane protein complex.